Genetic engineering has provided a method to isolate, selectively amplify, and express genes encoding desirable traits. These genes are often obtained from one organism and transformed into another organism so that expression of the gene can be manipulated and maximized. The transfer of genes from one organism to another can be used to produce large quantities of the gene product as well as to provide the transformed organism with improved characteristics or traits. Heterologous genes encoding desirable traits can be introduced into a wide variety of prokaryotic and eukaryotic hosts. For example, advantageous genes encoding herbicide resistance from bacteria can be incorporated into a plant's genome. The bacterial gene can then be expressed in the plant cell to confer on the plant cell resistance to the herbicide.
In order for the newly inserted gene to be expressed in a eukaryotic or prokaryotic host cell, proper regulatory sequences must be present and in the proper location with respect to the coding sequence. These regulatory sequences include a promoter region and a 3' nontranslated regulatory region. The promoter is a region of DNA sequences located upstream from the coding sequence of the gene. The function of a promoter sequence is to allow access and position of the transcription enzyme RNA polymerase in the vicinity of the transcription initiation site. The promoter DNA sequence can contain regulatory sequences that influence the rate and timing of the transcription of the gene. For example, insertion of a 21 base pair sequence into the 35S cauliflower mosaic virus (CaMV) promoter results in a tissue-specific expression in roots (Lam et al., PNAS, 86:7890 (1989).) Other sequences, like the TATA box and the CCAAT box in eukaryotic promoters, are known to influence the rate and level of gene transcription.
Certain promoters are known to be strong promoters. These promoters direct transcription at higher levels than other types of promoters and are capable of directing expression in other types of cells. One of the promoters that is a strong promoter in some types of plant cells is the 35S cauliflower mosaic virus (CaMV) promoter. However, the 35S CaMV promoter expression in plants can be variable and is especially so in monocotyledonous plants. Thus, strong promoters in one system often are not capable of providing for gene expression in a wide variety of both prokaryotic and eukaryotic host cells.
Chlorella viruses are a large group of recently identified viruses that infect certain eukaryotic green algae Chlorella. Chlorella viruses can be produced in large quantities and can be assayed by plaque formation. Chlorella viruses are large (150 to 190 nm) polyhedral plaque forming viruses containing greater than 300 kilobases of linear double-stranded DNA. The viruses are placed into 16 classes on the basis of plaque size antibody reactivity and the nature and abundance of methylated bases in their genomic DNA.
The Chlorella viruses have several unique features. The viruses have enough DNA sequence to encode 200 to 300 proteins. It is known that each virus contains and encodes 50 structural genes. The viruses also encode several DNA methyltransferase genes, DNA restriction endonuclease genes, and DNA polymerase genes. The DNA methyltransferase genes and the restriction endonuclease genes have been studied as a unique DNA restriction-modification system. Because the Chlorella viruses can be grown to large quantities and have several unique features, they are good candidates for the isolation of factors important in gene regulation.
Thus, there is a need for identifying and isolating strong promoters that are capable of expressing heterologous genes in a wide variety of cell types. There is also a need to identify and isolate strong promoters that can function in monocotyledonous plants, like wheat or rice. There is also a need to identify, isolate and characterize the promoters of the Chlorella virus genes including the DNA methyltransferase genes.